DUSP11 Antibody, FITC conjugated

What is DUSP11 and what are its primary functions?

DUSP11, also known as PIR1 (phosphatase interacting with RNA and ribonucleoprotein 1), is a 40-kDa dual specificity phosphatase belonging to the protein tyrosine phosphatase (PTP) family. DUSP11 functions primarily as an RNA phosphatase that regulates noncoding RNA stability . It can dephosphorylate tyrosyl-phosphorylated poly(GluTyr), serine/threonine residues, and RNA trinucleotides . Recent research has revealed that DUSP11 also targets proteins, particularly TGF-β–activated kinase 1 (TAK1), demonstrating its dual activity on both RNA and protein substrates . DUSP11 has been implicated in immune cell signaling, RNA metabolism regulation, and viral replication inhibition .

What is the subcellular localization of DUSP11?

DUSP11 primarily localizes to the nucleus, with approximately 90% of endogenous DUSP11 found in the nuclear fraction in A549 cells, while approximately 10% is present in the cytoplasm . This predominantly nuclear localization is consistent with its reported interactions with RNA splicing factors. Within the nucleus, DUSP11 has been shown to co-localize with SC35, a mammalian splicing factor, suggesting its involvement in RNA processing mechanisms .

What is the structure of the DUSP11 gene and how is it regulated?

The DUSP11 gene contains at least two putative p53 DNA binding sites in its promoter region, making it a p53 target gene . Expression of DUSP11 is induced in a p53-dependent manner following treatment with DNA damaging agents . Structurally, the gene contains multiple exons, with the targeting strategy for DUSP11-deficient mice involving insertion between exon 1 and exon 2 . The catalytic active site contains a critical cysteine at position 152 (Cys152), mutation of which completely abolishes its phosphatase activity .

What detection methods are optimal when using FITC-conjugated DUSP11 antibodies?

When using FITC-conjugated DUSP11 antibodies, flow cytometry and immunofluorescence microscopy are the optimal detection methods. For flow cytometry, a concentration of 0.5-1 μg antibody per 10^6 cells is typically recommended, with excitation at 488 nm and emission detection at 530/30 nm. For immunofluorescence, cells should be fixed with paraformaldehyde (typically 4%) and permeabilized with 0.1-0.5% Triton X-100, followed by blocking and antibody incubation (typically 1-5 μg/ml) . When performing co-localization studies, such as with nuclear splicing factors, confocal microscopy offers the best resolution to visualize nuclear DUSP11 distribution patterns .

How can I validate DUSP11 antibody specificity for research applications?

To validate DUSP11 antibody specificity, several approaches are recommended:

• Use of knockout models: Compare antibody binding between wild-type and DUSP11-deficient samples. DUSP11 knockout mice and cell lines have been successfully generated and characterized .

• Western blotting: Verify a single band at approximately 40 kDa with reduced or absent signal in DUSP11-deficient samples .

• Immunoprecipitation followed by mass spectrometry: This approach can confirm antibody specificity by identifying DUSP11 and its interacting partners in immunoprecipitates .

• Immunofluorescence comparison: Compare staining patterns between wild-type and DUSP11-deficient cells, with expected nuclear predominance in wild-type cells .

• Competitive binding assays: Pre-incubation with recombinant DUSP11 should diminish antibody signal if the antibody is specific.

What are the recommended protocols for in situ proximity ligation assay (PLA) using DUSP11 antibodies?

For in situ PLA to detect DUSP11 interactions with target proteins such as TAK1, the following methodology has been validated:

• Stimulate cells with appropriate ligands (e.g., LPS for macrophages) at defined time points.

• Deposit cells on glass slides and pretreat with fixation and blocking solutions.

• Incubate with primary antibodies against DUSP11 and the target protein (e.g., TAK1).

• Apply oligonucleotide (PLA Probe MINUS or PLA Probe PLUS)-conjugated secondary antibodies.

• Perform ligation and amplification reactions according to manufacturer's protocols (e.g., Duolink In Situ Red Starter Kit, Sigma-Aldrich).

• Wash slides and mount using a mounting medium containing DAPI.

• Analyze PLA signals from paired proteins in close proximity (<40 nm) .

This method has successfully demonstrated that DUSP11 interacts with TAK1 upon LPS stimulation in macrophages, with enhanced interaction following stimulation .

What proteins are known to interact with DUSP11 and how can these interactions be studied?

DUSP11 has been shown to interact with several proteins including:

To study these interactions, researchers typically employ:

• Coimmunoprecipitation: This approach has successfully demonstrated DUSP11 interactions with TAK1 but not TAB1 proteins, while related phosphatase DUSP14 showed the opposite interaction pattern .

• In situ proximity ligation assay (PLA): PLA has confirmed enhanced interaction between DUSP11 and TAK1 following LPS stimulation in macrophages .

• Mass spectrometry of immunocomplexes: Flag-tagged DUSP11 has been used to identify interaction partners by liquid chromatography-mass spectrometry after coimmunoprecipitation .

• Reciprocal coimmunoprecipitation assays: Using differently tagged proteins (e.g., Myc-DUSP11 and Flag-TAK1) to confirm bidirectional interactions .

How does DUSP11 regulate TAK1 phosphorylation and what are the functional consequences?

DUSP11 directly interacts with and dephosphorylates TAK1, a key mediator of TLR4 signaling. The DUSP11-TAK1 interaction is enhanced following LPS stimulation in bone marrow-derived macrophages (BMDMs) . Functionally:

• DUSP11 dephosphorylates TAK1 at the Ser412 residue, but this activity requires intact phosphatase function, as the catalytically inactive C152S mutant lacks this ability .

• DUSP11 deficiency enhances LPS-induced TAK1 phosphorylation in BMDMs, leading to increased production of inflammatory cytokines .

• In vivo, DUSP11-deficient mice show increased susceptibility to LPS-induced endotoxic shock and elevated cytokine production, demonstrating that DUSP11's regulatory effect on TAK1 has significant physiological consequences for immune responses .

This mechanism represents a novel discovery that DUSP11, previously known primarily as an RNA phosphatase, also functions as a protein phosphatase targeting a key immune signaling molecule .

How can DUSP11 antibodies be used to study its role in viral infection models?

FITC-conjugated DUSP11 antibodies can be valuable tools in studying DUSP11's role in viral infection models through several approaches:

• Tracking DUSP11 relocalization during viral infection: Using fluorescence microscopy, researchers can monitor changes in subcellular localization of DUSP11 during infection with viruses such as HCV .

• Flow cytometric analysis in infected cells: Quantitative assessment of DUSP11 expression levels in infected versus uninfected cells can be performed using flow cytometry .

• Dual labeling with viral markers: Co-localization studies using FITC-DUSP11 antibodies alongside antibodies against viral proteins can reveal potential interactions or proximity during infection .

• Time-course experiments: DUSP11 expression and localization can be monitored at different stages of viral infection to understand temporal regulation .

Research has shown that DUSP11 inhibits HCV replication, as DUSP11 knockout cells show significantly increased HCV replicon activity compared to parental cells . Similar inhibitory effects have been observed with Orsay virus replication, likely through an RNAi-based mechanism . These studies demonstrate DUSP11's importance in antiviral responses.

What is known about the role of DUSP11 in RNA processing and how can this be investigated?

DUSP11 plays multiple roles in RNA processing, particularly affecting miRNA biogenesis and function:

• DUSP11 dephosphorylates 5'-triphosphorylated RNAs: This activity enables subsequent exonuclease processing by enzymes like XRN .

• DUSP11 affects noncanonical miRNA silencing: It alters the silencing potential of noncanonical viral miRNAs in mammalian cells by promoting incorporation of 5p miRNAs into RISC .

• DUSP11 is involved in RNAi pathways: In C. elegans, DUSP11 suppresses Orsay virus replication through RNAi-based mechanisms, interacting with Dicer and ERI-1 complexes .

To investigate these functions, researchers can:

• Utilize DUSP11 antibodies in RNA-immunoprecipitation (RIP) experiments to identify associated RNA species

• Perform CLIP-seq (cross-linking immunoprecipitation followed by sequencing) to map DUSP11-RNA interactions genome-wide

• Analyze miRNA processing and RISC loading in DUSP11 wild-type versus knockout cells using northern blotting and small RNA sequencing

• Examine co-localization with RNA processing bodies using immunofluorescence microscopy

These approaches have revealed that DUSP11's RNA phosphatase activity specifically affects 5'-triphosphorylated RNAs but not canonically processed miRNAs .

What are common issues when using DUSP11 antibodies and how can they be resolved?

When performing co-localization studies with DUSP11 and its interacting partners (such as TAK1 or splicing factors), careful optimization of fixation and permeabilization conditions is essential, as over-fixation can mask epitopes and under-permeabilization can limit antibody access to nuclear DUSP11 .

How can I optimize DUSP11 detection in primary immune cells?

For optimal detection of DUSP11 in primary immune cells, particularly macrophages and lymphocytes, consider the following protocol optimizations:

• Cell preparation: Fresh isolation is preferred for primary cells, with minimal manipulation before fixation to preserve native protein states and localization .

• Fixation and permeabilization: For bone marrow-derived macrophages (BMDMs), 4% paraformaldehyde for 15 minutes followed by 0.1% Triton X-100 for 10 minutes has proven effective .

• Blocking: Extended blocking (1-2 hours) with 5% serum from the same species as the secondary antibody helps reduce background, particularly important in primary cells which may have higher autofluorescence .

• Antibody concentration: Primary cells often require higher antibody concentrations (typically 2-5 μg/ml) compared to cell lines .

• Signal amplification: Consider tyramide signal amplification systems for low-abundance detection in primary cells.

• Controls: Include DUSP11-deficient primary cells as negative controls to confirm specificity .

This approach has successfully been used to detect DUSP11-TAK1 interactions in BMDMs using proximity ligation assays .

How should results from DUSP11 knockout models be interpreted in relation to immune function?

When interpreting results from DUSP11 knockout models in immune function studies, several key considerations should be kept in mind:

• Normal lymphoid development: DUSP11-deficient mice show normal development of CD4⁻CD8⁻ double-negative, CD4⁺CD8⁺ double-positive, CD8⁺ single-positive, or CD4⁺ single-positive thymocytes. They also display similar proportions of CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, and B220⁺ B cells in the spleen and lymph nodes compared to wild-type mice . This indicates that DUSP11 is not essential for immune cell development.

• Enhanced LPS responses: DUSP11-deficient macrophages show enhanced TAK1 phosphorylation and increased cytokine production following LPS stimulation . This suggests DUSP11 functions as a negative regulator of TLR4 signaling.

• Increased susceptibility to endotoxic shock: DUSP11-deficient mice are more susceptible to LPS-induced endotoxic shock in vivo, demonstrating physiological relevance of DUSP11's regulatory function in inflammatory responses .

• Potential compensation: When interpreting negative results (lack of phenotype in certain pathways), consider potential compensation by other DUSP family members, particularly in developmental contexts .

• Cell type-specific effects: DUSP11's regulatory role may vary across different immune cell types, requiring careful interpretation when using pan-immune knockout models .

What controls are essential when performing co-localization studies with DUSP11 antibodies?

When conducting co-localization studies using FITC-conjugated DUSP11 antibodies, the following controls are essential:

• Antibody specificity controls:

  • DUSP11 knockout or knockdown cells to confirm absence of specific signal

  • Isotype control antibodies matched to the primary antibody

  • Competitive blocking with recombinant DUSP11 protein

• Fluorophore controls:

  • Single fluorophore controls to establish bleed-through parameters

  • Unstained samples to determine autofluorescence levels

  • Secondary antibody-only controls to detect non-specific binding

• Biological controls:

  • Positive controls where co-localization is expected (e.g., DUSP11 with nuclear splicing factors)

  • Negative controls where co-localization is not expected (e.g., DUSP11 with mitochondrial markers)

  • Treatment controls that alter localization (e.g., pre- and post-LPS stimulation for DUSP11-TAK1 interaction)

• Technical controls:

  • Z-stack imaging to confirm true co-localization versus superimposition

  • Quantitative co-localization analysis using appropriate coefficients (Pearson's, Manders', etc.)

  • Fixed imaging parameters across all experimental conditions

Implementing these controls has allowed researchers to definitively demonstrate enhanced interaction between DUSP11 and TAK1 following LPS stimulation using in situ proximity ligation assays .

What are the emerging research areas involving DUSP11 in disease models?

Recent advances in DUSP11 research have revealed its potential significance in several disease models:

• Inflammatory disorders: Given DUSP11's role in negatively regulating TAK1 phosphorylation and subsequent inflammatory responses, DUSP11-targeting strategies could offer therapeutic potential for inflammatory diseases . DUSP11-deficient mice show increased susceptibility to LPS-induced endotoxic shock, suggesting DUSP11 activators might dampen excessive inflammation .

• Viral infections: DUSP11 has demonstrated antiviral activity against HCV and Orsay virus through its effects on RNA processing and RNAi pathways . In HCV models, DUSP11 knockout cells show enhanced viral replicon activity, suggesting DUSP11 as a potential antiviral target .

• Cancer biology: As a p53 target gene, DUSP11 may play a role in tumor suppression . Ectopic expression of wild-type DUSP11, but not catalytically inactive mutants, leads to growth arrest in colony formation and proliferation assays . Conversely, inhibition of DUSP11 expression by shRNA increases the proliferation of normal and DNA-damaged cells in tissue culture .

• RNA processing disorders: DUSP11's involvement in RNA splicing through interactions with factors like SAM68 suggests potential roles in diseases characterized by splicing dysregulation .

Researchers are now exploring these disease connections using DUSP11 antibodies to characterize expression patterns in patient samples and disease models.

How can multiplex imaging approaches be optimized when using FITC-conjugated DUSP11 antibodies?

For optimal multiplex imaging using FITC-conjugated DUSP11 antibodies alongside other fluorescent markers:

• Spectral compatibility planning:

  • FITC (excitation ~495 nm, emission ~520 nm) pairs well with far-red fluorophores (e.g., Cy5, Alexa Fluor 647)

  • Avoid spectrally overlapping fluorophores like TRITC or YFP

  • Consider spectral unmixing for closely overlapping signals

• Sequential staining protocols:

  • When antibodies are raised in the same species, perform sequential staining with complete blocking between steps

  • Direct conjugates minimize cross-reactivity in multiplex applications

• Validated multiplex combinations:

  • DUSP11-FITC + TAK1 (far-red) for immune signaling studies

  • DUSP11-FITC + nuclear splicing factors (red) for RNA processing research

  • DUSP11-FITC + viral proteins (far-red) for infection models

• Advanced imaging approaches:

  • Consider cyclic immunofluorescence for highly multiplexed imaging

  • Spectral imaging with linear unmixing can resolve closely overlapping fluorophores

  • Super-resolution microscopy (STORM, PALM) for detailed co-localization studies

• Image acquisition optimization:

  • Sequential channel acquisition to minimize bleed-through

  • Consistent exposure settings between samples

  • Appropriate nuclear counterstain (DAPI works well with FITC)

These approaches have been successfully applied to visualize DUSP11 interactions with multiple binding partners and its subcellular localization patterns in various experimental contexts .